The principal investigator's interest has been in the developmental aspects of electrolyte transport across the intestinal epithelium. Initially, the PI.. utilized in vivo perfusion and in vitro plasma membrane vesicles to define the transport processes of Na+ and Cl transport across the enterocytes. The developmental pattern of these processes, and their regulation was defined. The current proposal will extend knowledge acquired during the tenure of the grant to explore and identify the intestinal Na+/H+ exchangers at the brush border and basolateral membranes of the enterocyte of the rat and their regulation during development. The brush border Na+/H+ exchanger functions as part of the neutral Na+/Cl transport process; whereas, the basolateral exchanger functions as a regulator of intracellular Ph and cell volume. We have shown developmental changes in the transport characteristics of both exchangers in regard to their kinetics and amiloride sensitivity. Moreover, the amiloride sensitivity of both exchangers is markedly deferent indicating encoding by two separate genes. The specific aims of the proposal are; 1) Clone and sequence the full length rat cDNA encoding the brush border and basolateral Na+/H+ exchanger using probes derived from human Na+/Ha+ exchanger cDNA.2) determine chromosomal assignment of both exchangers and whether they are encoded by two separate genes. 3. Determine the chromosomal localization of the Na+/H= exchanger loci by genetic linkage analysis and identify genetic markers (Restriction fragment length polymorphism [RFLPs] and Polymerase Chain Reaction amplification product RFLP's for use in studies of infants with defect in the brush border Na+/H+ exchanger. 40 Express the brush border and basolateral Na+/H+ exchanger in xenopus Laevis oocytes and in exchanger deficient cells. 50 Determine the tissue distribution of Na=/H+ exchangers expression during maturation (suckling, weanling, and adolescent rats) and expression along villus-crypt axis by Northern blotting and in situ hybridization using cDNA'S of both exchangers as probes. 6) Further localize the site of Na+/H= exchange expression by Western blotting and immunocytochemistry using polyclonal antibodies against both exchangers. 7) Determine if both exchangers are phosphoproteins and whether second messengers affect their phosphorylation. 8) Determine the structure- function relationship of both exchangers.